Rhamnolipids (RLs) are interface-active glycolipids produced by various bacterial species. RLs consist of one (monorhamnosylipids or mono-rhamnolipids) or two rhamnose units (dirhamnosylipids or di-rhamnolipids) and one or two (predominantly two) 3-hydroxy fatty acid residues. Rhamnolipids have particular surface-active properties such as, for example, a strong foaming ability, and are of interest for highly diverse technical applications, particularly as surfactants (reviewed in Randhawa et al. (2014) “Rhamnolipid biosurfactants—past, present, and future scenario of global market”, Frontiers in Microbiology 5:1-7; Muller et al. (2012) “Rhamnolipids—Next generation surfactants?”, J Biotechnol 162(4):366-80; Banat et al. (2010) “Microbial biosurfactants production, applications and future potential” Appl Microbiol Biotechnol 87:427-444). Examples of potential applications include: (1) bioremediation and enhanced oil recovery (EOR) since they efficiently remove crude oil and heavy metals from contaminated soil and facilitate bioremediation of oil spills due to their emulsification properties; (2) cosmetics: including cosmeceuticals for example for wound healing, burns, psoriasis and wrinkles; (3) detergents and cleaners: particularly for laundry products, shampoos and soaps given their surface active and emulsification properties and (4) agriculture. Rhamnolipids are currently available in an agricultural anti-fungal product marketed as ZONIX™ (Proptera LLC).
Interest in RLs has increased since they can be prepared by means of fermentation based on renewable raw materials. However, rhamnolipids have only been available on the market in small amounts and at high prices partially due to cumbersome downstream processing methods. Various methods have been disclosed for producing rhamnolipids (reviewed in, for example, Heyd et al. (2008) “Development and trends of biosurfactant analysis and purification using rhamnolipids as an example”, Anal Bioanal Chem 391:1579-1590 and Smyth et al. (2010) “Isolation and Analysis of Low Molecular Weight Microbial Glycolipids”, in Handbook of Hydrocarbon and Lipid Microbiology, K. N. Timmis (ed.), Springer-Verlag, Berlin, pp. 3705-3724; Desai et al. (1997) “Microbial Production of Surfactants and Their Commercial Potential”, Microbiol. Mol. Biol. Rev. 61: 47-64 and also disclosed in for example U.S. Pat. No. 5,656,747, U.S. Pat. No. 4,628,030, US20140148588, CN102796781, CN102766172, CN101787057, CN101845468, CN102432643, CN1908180, KR1020060018783).
Currently, the most common isolation procedure for rhamnolipids involves autoclaving fermentation broth right after fermentation is complete, followed by acidifying to precipitate rhamnolipids out. The solid rhamnolipids that result are contaminated with solid cellular material from the organism used in the fermentation. RLs are isolated from these cellular solids by extracting them into an organic solvent such as ethyl acetate. After stripping the ethyl acetate, a concentrated oily form of the product results. However, the solvent extraction process brings along any hydrophobic impurities with it, potentially including unconsumed triacyl glyceride oil and antifoam from the fermentation process. Examples of other methods disclosed include (1) aluminum sulphate precipitation of fermentation broth followed by extraction with organic solvent; (2) continuous ultrafiltration; (3) column chromatography (see, for example, Lebrón-Paler A (2008) “Solution and interfacial characterization of rhamnolipid biosurfactant from P. aeruginosa ATCC 9027” PhD Dissertation University of Arizona, CN101787057, CN101407831, CN1908180) using either adsorption, ion exchange, reversed phase or normal phase columns which may be performed in combination with the precipitation step; (4) countercurrent chromatography (Zhang et al. “Separation and purification of six biosurfactant rhamnolipids by high-speed countercurrent chromatography utilizing novel solvent selection method”, Separation Science and Technology, in press (posted Nov. 24, 2015)); (5) selective crystallization followed by extraction or (6) foam fractionation/adsorption (see, for example, US 2015/0011741; Sarachat et al., (2010) “Purification and concentration of a rhamnolipid biosurfactant produced by Pseudomonas aeruginosa SP4 using foam fractionation”, Bioresource Technology 101: 324-330). However, all of these methods have various disadvantages as well (e.g., expensive, do not scale well, resource intensive, etc.).